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Applications
of Phase Contrast PHASE CONTRAST MICROSCOPY Living cells and most cell organelles are often difficult if not impossible to see by brightfield microscopy because they do not absorb, refract or diffract sufficient light to contrast with the surrounding medium. The phase contrast microscope was developed to improve contrast differences between cells/organelles and the surrounding medium, making it possible to see cells/organelles without staining. The technique is based on the fact that cells differ in refractive index from their surroundings and thus bend some of the light rays passing through them. Light rays passing through a transparent specimen (most unstained cells are transparent) emerge as either direct rays unaffected by passage through the specimen (unaltered in intensity and phase) or diffracted rays bent as they pass through the specimen (altered intensity and/or phase). This effect is amplified by the phase annulus coupled with special phase rings in the objectives leading to a dark image on a light background. Click here to view samples of phase contrast microscopy. FLUORESCENT MICROSCOPY The fluorescent microscope is used to visualize specimens that fluoresce, that is, emit light of a different color (always a higher wavelength) than the light absorbed by the specimen. Fluorescence occurs after light is absorbed either because of naturally occurring fluorescent materials in the cells (such as chlorophyll and related molecules) or because the cells have been stained with a fluorochrome. Fluorochromes are stains similar to cell and tissues stains used in light microscopy and have been chosen or designed to be highly specific in their attachment to molecules in cells. The use of fluochromes has made it possible to identify cells and subcellular components of cells with a high degree of specificity amid non-fluorescing material. Our Olympus
microscope is equipped with 3 filter cubes: FITC, TRITC and DAPI, named
for the common dyes they are used with.
* excitiation (Ex) and emission (Em) wavelengths These filter cubes are also used with many other fluorochromes. Click here to view other applications for the named filter cubes (fluorescence mirror units). |